The long-term goal of this project is to define molecular mechanisms that control synapse assembly and growth. Drosophila NMJ is a glutamatergic synapse, similar in structure and physiology to mammalian central excitatory synapses. In flies each NMJ is unique and identifiable, synapses are large and accessible for electrophysiological and optical analysis, making the Drosophila NMJ a powerful genetic system to study synapse development. The Drosophila NMJ can thus be used to analyze and model defects in the structural and physiological plasticity of glutamatergic synapses, which are associated with a variety of human pathologies from learning, memory deficits to autism. The similarity in gross architecture, function, and molecular machinery supports the notion that studying the assembly and development of fly glutamatergic synapses will shed light on their vertebrate counterparts. In flies the subunits that form the glutamate-gated ion channels (iGluRs) are known and relatively well studied. However the mechanisms that control iGluRs clustering and stabilization at the postsynaptic densities, a key step that confers functionality to the nascent synapse, remain a mystery. Several mechanisms have been identified that regulate the subunit compositions and the extent of iGluRs synaptic localization, but no molecules other than the receptors themselves were shown to be absolutely required for clustering of the receptor complexes. We have recently discovered that the Neuropillin and Tolloid-like protein, Neto, is an essential component for the clustering of the iGluRs at the Drosophila NMJ. Neto belongs to a family of highly conserved proteins sharing an ancestral role in formation and modulation of glutamatergic synapses. Two vertebrate homologs called Neto-1 and -2, have been recently characterized and shown to modulate the properties of selective iGluRs. Neto1/Neto2 double knockout mice are viable but have defects in long-term potentiation, and in learning and memory. To study the function of Drosophila Neto, we generated an allelic series and performed behavioral, anatomical, biochemical and physiological analyses. We found that Neto is absolutely required for trafficking and clustering of iGluRs at Drosophila NMJ. Neto directly engages the iGluRs in complexes that are targeted and stabilized at synaptic sites. Furthermore, Neto and iGluRs depend on each other for targeting and clustering at the NMJ at the onset of synaptogenesis. Our studies indicate that Neto 1) engages the iGluR complexes, 2) regulates iGluRs synaptic targeting and clustering, and 3) stabilizes the mature PSDs. Optimal Neto appears crucial for synapse assembly since genetic manipulations of Neto levels shift iGluRs distribution to extrajunctional locations, without altering iGluRs expression. In addition, Neto may modulate the iGluRs channel properties. Neto does not contain any catalytic domain; instead, it has a number of extracellular protein interaction domains, and an intracellular domain rich in putative phosphorylation sites and docking motifs. Therefore, Neto functions in iGluRs synaptic targeting and PSD stabilization likely via binding to iGluRs and/or other interaction partners. On-going structure-function analyses are focused on mapping the known functions of Neto to individual domains of the protein. Preliminary studies indicate that the intracellular domain of Neto is required for regulation of synaptic targeting. Furthermore, Drosophila neto locus encodes for two isoforms generated by alternative splicing that differ in their intracellular domains. Both intracellular domains contain multiple putative phosphorylation sites raising the possibility of rich modulation of Neto/iGluRs distribution in response to the muscle status.